Project Summary/Abstract Drug-resistant tuberculosis is a global public health crisis. Mycobacterium tuberculosis (Mtb) causes ~1.3 million deaths each year. 5% of new Mtb infections are drug-resistant, driving an urgent clinical need for new anti-Mtb therapeutics. This proposal supports efforts to combat drug-resistant Mtb by dissecting the function and pharmacological dysregulation of Clp proteases, a family of novel antibacterial targets. Clp proteases harness chemical energy to destroy folded proteins in the cytosol of bacteria. Clp proteases are essential in mycobacteria, and compounds that disrupt their function can kill Mtb and potentially treat drug-resistant tuberculosis. Our prior work on Mtb Clp proteases revealed distinctive characteristics that set them apart from well-studied homologs. We hypothesize that mycobacteria exploit these characteristics to regulate Clp protease assembly and activity across stages of mycobacterial growth and infection. However, no studies have yet examined how these features contribute to mycobacterial physiology, and this deficiency in our understanding hampers efforts to develop Clp- targeting therapeutics. This sub-project aims to addresses outstanding questions surrounding Mtb Clp protease regulation, and to develop compounds optimized to target Mtb Clp proteases. In Aim 1, we will interrogate Clp protease regulation by investigating how and when Clp proteases form proteolytically active complexes. We will use biophysical approaches to define the kinetic trajectory of assembly, and identify active and inactive complex intermediates that form along the assembly pathway. Additionally, we will use a model mycobacterial system to correlate assembly state and activity state within the cell. Understanding these details will improve our ability to effectively target these enzymes during infection. In Aim 2, we seek to elaborate existing chemical scaffolds to create new compounds optimized to target Mtb Clp enzymes. We will develop novel probes that modulate or report on Clp protease assembly, and new lead compounds with improved affinity and target selectivity. Structural information, together with our existing toolbox of biochemical assays, will guide compound development and will be used to evaluate compound affinity and selectivity. We will also create and screen a compound library, derived from an existing scaffold, to identify novel leads. Together, these studies will expand our understanding of Mtb Clp protease function, and support the development of therapeutics capable of combating drug-resistant tuberculosis.